Compositions for treating corns, calluses and warts

ABSTRACT

Topical compositions for the treatment of corns, calluses and warts comprising a benzenediol or a substituted 1,2-benzenediol and a pharmaceutically acceptable carrier, are described.

The present application is the United States national applicationcorresponding to international Application No. PCT/US 94/08315, filedAug. 11, 1994 and designating the United States, which PCT applicationis in turn a continuation-in-part of U.S. application Ser. No.08/107553, filed Aug. 17, 1993, the benefit of which applications areclaimed pursuant to the provisions of 35 U.S.C. 120, 363 and 365 (C).

BRIEF SUMMARY OF THE INVENTION

The invention relates to topical compositions for treating corns,calluses, and warts which comprise a benzenediol. Included amongbenzenediols are benzenediols or substituted benzenediols selected fromthe group consisting of hydroquinone, olivetol, pyrocatechol,2,5-dihydroxy benzoic acid; or 1,2-benzenediols substituted in the4-position selected from the group consisting of nordihydroguaiareticacid (NDGA) and 4-nitrocatechol, in combination with a pharmaceuticallyacceptable carrier. The invention also relates to a method for treatingthe above-described conditions which comprises administering aneffective amount of a benzenediol or substituted benzenediol selectedfrom the group consisting of hydroquinone, olivetol, pyrocatechol, and2,5-dihydroxy benzoic acid or 1,2-benzenediols substituted, for example,on the 4-position, such as nordihydroguaiaretic acid (NDGA) and4-nitrocatechol.

DETAILED DESCRIPTION OF THE INVENTION

Hyperkeratotic tissues, such as: corns (heloma), calluses (tyloma) andwarts (condyloma), are well defined, thickened lesions of the epidermis.They occur at skin sites that are normally involved in chronicmechanical stress (corns and calluses) or infected with papilloma virus(warts). Pain produced by the thickened tissue can cause these lesionsto be debilitating.

Traditionally, "keratolytic agents", such as: salicylic acid andresorcinol, have been applied topically to these lesions to solubilizeintercellular bonds resulting in desquamation of the thickened,hyperkeratotic tissues.

The goal was to develop a faster acting corn and callus remover product.To achieve this goal benzenediols were evaluated. Assays of keratinocytedifferentiation and keratolytic action, as described below were employedto identify the compounds that possess this activity.

As used herein, "alkyl" means a straight or branched chain alkyl group.Alternatively, the number of carbon atoms in a particular alkyl groupmay be specified. For example, C₁ -C₈ alkyl, refers to a straight orbranched chain alkyl group having one to eight carbon atoms. Halo refersto chloro, bromo, iodo, and fluoro.

The present invention provides new topical compositions for thetreatment of all kinds of corns, calluses, or warts. The compositions ofthe invention provide for faster removal of corns, calluses, and wartsthan do prior art compositions. The compositions of the invention canalso be used in the treatment of hyperkeratinizing andhyperproliferative skin diseases and conditions such as ichthyoses,porokeratoses, follicular keratoses, palmoplantar keratodermas,psoriasis, eczema, dandruff and dry skin. The invention also relates toa method for treating the above-described medical conditions whichcomprises administering an effective amount of a benzenediol orsubstituted benzenediol selected from the group consisting ofhydroquinone, olivetol, pyrocatechol, 2,5-dihydroxy benzoic acid; or a1,2-benzenediol substituted in the 4-position selected from the groupconsisting of nordihydroguaiaretic acid (NDGA) and 4-nitrocatechol.

The topical compositions of the present invention comprise abenzenediol. As used herein, "benzenediol" means the following: abenzenediol or a substituted benzenediol. Among substituted benzenediolsare dihydroxyphenylalkyl benzenediols. Exemplary of benzenediols andsubstituted benzenediols are compounds such as hydroquinone, olivetol,pyrocatechol, 2,5-dihydroxy benzoic acid; or a 1,2-benzenediolsubstituted, for example, in the 4-position. More specifically, thesubstituents which can occur in the 4-position or in other positions onthe benzene in the compounds of the invention are nitro, halo, C₁ -C₈alkyl, or dihydroxyphenyl-C₁ -C₈ alkyl-. Specific 1,2-benzenediolssubstituted in the 4-position include nordihydroguaiaretic acid (NDGA)and 4-nitrocatechol.

A preferred benzenediol of the invention is pyrocatechol.

Pyrocatechol is a 1,2-benzenediol. The chemical structure ofpyrocatechol is as follows: ##STR1##

Pyrocatechol is a crystalline compound with a phenolic odor.Pyrocatechol has several industrial applications and has been used inthe past as a topical antiseptic (Merck Index, 11th Edition, 1989, S.Budavari, Editor, Merck and Company, Rahway, N.J., page 1272). Theantiseptic properties of pyrocatechol derive from its relatedness tophenol. Pyrocatechol has been used in hair dye at concentrations of ≦1%(Final Report on the Safety Assessment of Hydroquinone and Pyrocatechol,1986, Journal of American College of Toxicology, 5:123-165).

A more preferred 1,2-benzenediol substituted in the 4-position isnordihydroguaiaretic acid (NDGA), a substituted 1,4-benzenediol, whichhas the following structure: ##STR2##

NDGA is a crystalline, odorless powder. It is used as an antioxidant infats and oils (Merck Index, 11th Edition, 1989 S. Budavari, Editor,Merck Company, Rahway N.J., Pages 1057-1058).

The topical compositions of the invention also comprise a pharmaceuticalcarrier material suitable for topical use.

A composition in accordance with the invention comprises a benzenediolin a range of about 2 to about 64% (weight/weight).

More specifically, the topical compositions of the invention contain abenzenediol or substituted benzenediol selected from the groupconsisting of hydroquinone, olivetol, pyrocatechol, 2,5-dihydroxybenzoic acid; or a 1,2-benzenediol substituted in the 4-positionselected from the group consisting of nordihydroguaiaretic acid (NDGA)and 4-nitrocatechol, at a range of about 2 to about 64% (weight/weight).A preferred range for pyrocatechol or NDGA is about 3 to about 24%(weight/weight). As used herein, the term "weight/weight" means theratio of the weight of the particular ingredient in question to theweight of the entire composition.

Those skilled in the art will be able to maximize the safety andefficacy of a given formulation. Compositions of the invention can takeany of the following delivery forms: salves, lotions, plaster devices,collodion-type vehicles, suspensions, ointments, creams, gels, sprays,bandages, patches or other appropriate topical vehicles or deliverydevices.

Topical compositions of the invention contain a pharmaceuticallyacceptable carrier. The pharmaceutically acceptable carrier delivers theactive ingredient to the site of application. The pharmaceuticallyacceptable carrier may be a liquid such as glycol, petrolatum, ethanol,acetone, dimethyl sulfoxide (DMSO), and the like. The pharmaceuticallyacceptable carrier may also be pad devices, disks or plaster. Thepharmaceutically acceptable carrier may also be a film former such asflexible collodion, USP.

Topical compositions of the invention may also contain a viscosityenhancer. Viscosity enhancers increase the viscosity of the compositionso that it does not spread beyond the site of application. An example ofa viscosity enhancer is Balsam Fir (Oregon).

Topical compositions of the invention may also contain a film former.When a film former dries, it forms a protective film over the site ofapplication to prevent removal of active ingredient from the site. Anexample of a film former which may be used is Flexible Collodion,USPTopical compositions of the invention may also contain a colorantsuch as β-Carotene. Topical compositions of the invention may alsocontain a solvent which serves to dissolve the active ingredient. Anexample of a solvent which may be used is acetone. As can be seen, thesolvent may also sometimes serve as the carrier.

In preparing topical compositions of the invention, there can be addedconventional adjuvants such as propionic acid, propylene glycol, acetoneand lactic acid, conventional penetration enhancers such as erucic acid,oleic acid, and behenic acid; conventional buffers, preservatives,hydrophilic emulsifiers, lipophilic emulsifiers, sun-screening agents,perfumes, emollients, deodorants, humectants, and the like. Colorantsmay also optionally be added in the compositions of the invention.

Current collodion-based FDA monograph approved formulas may be employedin such topical liquid compositions.

One skilled in the art, would be able to devise other suitable liquidformulations.

Alternatively, current plaster pad-type FDA monograph approved formulasmay be used in devising compositions of the invention.

One skilled in the art would be able to devise a variety of suitableplaster pad-type formulations. Modified FDA monograph approved paddevices, disks or plaster may also be used as the carrier material. Oneskilled in the art would be able to apply benzenediols to these paddevices, disks or plaster to form a composition of the invention.

Modified FDA monograph approved liquid vehicles may be used as thecarrier material.

In preparing topical compositions of the invention, there can be addedconventional adjuvants such as propionic acid, salicylic acid, propyleneglycol, acetone and lactic acid, conventional penetration enhancers suchas erucic acid, oleic acid, and behenic acid; conventional buffers,preservatives, hydrophilic emulsifiers, lipophilic emulsifiers,sun-screening agents, perfumes, emollients, deodorants, humectants, andthe like.

In applying liquid formulations to the patient in need of suchtreatment, liquid formulations are applied, rubbed or spread on theaffected area of the skin. In applying plaster-pad formulations to thepatient in need of such treatment, plaster-pad formulations are appliedto the affected area of the skin so that the pad adheres to the skin.

In preparing topical compositions of the invention which contain NDGA,there can be added conventional adjuvants such as propionic acid,salicylic acid, propylene glycol, acetone and lactic acid, conventionalpenetration enhancers such as erucic acid, oleic acid, and behenic acid;conventional buffers, preservatives, hydrophilic emulsifiers, lipophilicemulsifiers, sun-screening agents, perfumes, emollients, deodorants,humectants, and the like. Colorants may also optionally be added in thecompositions of the invention.

Compositions of the invention are to be applied in a therapeuticallyeffective amount. A "therapeutically effective amount" means any amountwhich will cause improvement in a disease condition (such as removal ofa callus) when applied to the affected area repeatedly over a period oftime. The amount will vary with the condition being treated and theconcentration of the active ingredients in the formulation beingapplied. Appropriate amounts in any given instance-will be readilyapparent to those skilled in the art by routine experimentation.

Experiments were carried out which demonstrate that when a benzenediolsuch as pyrocatechol or NDGA is included in compositions, that theresulting composition is more effective in causing callus sloughing fromguinea pig footpads, than salicylic acid alone. These experiments aredescribed below.

Action of Pyrocatechol, NDGA or SA in Removal of Guinea Pig FootpadCallus: Description of Guinea Pig Footpad Assay (GPFA)

To evaluate keratolytic agents an animal model, GPFA, was developed. Theanalysis was performed according to the following protocol, using theguinea pig footpad as the test site. Three Hartley guinea pigs (250-400g) were used per test group for in vivo evaluations. Either right orleft footpads were treated with the opposite footpad as untreatedcontrol. For each treatment group, the same footpad was treated on eachanimal. The treated footpad received 200 μl of the test materialsaturated into a small cotton pledget. The cotton pledget was occludedwith several wrappings of Blenderm tape (3m Corporation, St. Paul,Minn.) and finally secured from removal with a wrapping of Zonas tape(Johnson and Johnson, New Brunswick, N.J.). Each group of three animalswas placed in a polycarbonate cage with contact bedding for 18-24 hours(overnight). At the end of the treatment period, the bandages wereremoved with surgical scissors and the footpads were examined for grosskeratolytic effects or dermatotoxicity. Observations were carried out atdaily intervals for one week. Clinical grades were recorded for possiblekeratolytic effects beginning on Day 2 and continuing through Day 7.Appropriate vehicle controls and bandage control groups were included ineach experiment. Pyrocatechol (Sigma Chemical Company, St. Louis, Mo.)and NDGA (Aldrich Chemical Co., Milwaukee Wis.) were prepared as a 12%(weight/weight) solution as described below. SA was prepared as a 12.6%(weight/weight) solution as described below.

    ______________________________________                                        Liquid Formulas for Evaluating Pyrocatechol and NDGA in the                   GPFA.                                                                                            Percentage of Formula                                      Component          % (weight/weight)                                          ______________________________________                                        Pyrocatechol or NDGA                                                                             12.0                                                       Balsam Fir (Oregon)                                                                              5.0                                                        β-Carotene, 22% in Vegetable Oil                                                            0.05                                                       Acetone            10.0                                                       Flexible Collodion, USP                                                                          q.s. 100.0                                                 ______________________________________                                    

The pyrocatechol formula in the preferred vehicle was prepared asfollows:

1) Balsam fir (Oregon) was mixed with acetone in a suitable vessel.

2) β-Carotene, 22% in vegetable oil, was added to the solution preparedin step 1 and thoroughly mixed.

3) Flexible collodion, USP, was added to the solution prepared in step 2and thoroughly mixed.

4) Pyrocatechol was added to the solution prepared in step 3.

5) The composition from step 4 was thoroughly mixed until thepyrocatechol was completely dissolved.

6) The composition made in step 5 was then transferred to appropriatecontainers for storage at room temperature ranging from about 22° toabout 27° C.

NDGA was formulated in the preferred vehicle in the same manner aspyrocatechol shown just above.

    ______________________________________                                        Liquid Formula for Evaluating SA in the GPFA.                                                    Percentage of Formula                                      Component          % (weight/weight)                                          ______________________________________                                        SA                 12.6                                                       Balsam Fir (Oregon)                                                                              5.0                                                        β-Carotene, 22% in Vegetable Oil                                                            0.05                                                       Acetone            10.0                                                       Flexible Collodion, USP                                                                          q.s. 100.0                                                 ______________________________________                                    

1) Balsam fir (Oregon) was mixed with acetone in a suitable vessel.

2) β-Carotene, 22% in vegetable oil, was added to the solution preparedin step 1 and thoroughly mixed.

3) Flexible collodion, USP, was added to the solution prepared in step 2and thoroughly mixed.

4) SA was added to the solution prepared in step 3.

5) The composition from step 4 was thoroughly mixed until thepyrocatechol was completely dissolved.

6) The composition made in step 5 was then transferred to appropriatecontainers for storage at room temperature ranging from about 22° toabout 27° C.

The relative efficacy of keratolytic action of the compounds for removalof guinea pig foot pad callus was determined by the following clinicalgrading scale:

    ______________________________________                                        Clinical Grade                                                                         Appearance of Footpad and Footpad Callus                             ______________________________________                                        0 =      No visible difference, smooth feet, or equivalent to                          control.                                                             0.5 =    Slight fine cracks in skin visibly different from control.           1 =      Somewhat larger cracks with edges turned up slightly.                2 =      Obvious separation of stratum corneum (SC) over a                             limited area of the footpad.                                         3 =      Separation of SC over a large area of the footpad.                   4 =      SC has peeled off completely revealing intact underlying                      epidermis, i.e., normal appearing skin.                              ______________________________________                                    

The clinical grades were then used to calculate a keratolytic efficacyscore referred to as the Keratolytic Index (KI). The following formulais used to calculate the KI for a given test group. ##EQU1##

The KI scores achievable for this analytical method range from 0, noapparent keratolytic activity, to 20, maximum keratolytic action. Theresults of a typical GPFA comparing the KI of pyrocatechol with that ofSA is given in Table 1 below. Pyrocatechol KI (11.1) and NDGA KI (6.7)both have a higher index score than salicylic acid (4.4) under thesetest conditions. The average KI in control groups was less than 1. Therewas no evidence of dermatotoxicity caused by pyrocatechol, NDGA or SA inany of the assays that were conducted.

                                      TABLE 1                                     __________________________________________________________________________    Table 1: Kl for Pyrocatechol, NDGA and SA Determined in the GPFA.             Treatment                                                                              Average Daily Clinical Grades                                        Group  (N)                                                                             Day-2                                                                              Day-3                                                                             Day-4                                                                              Day-5                                                                             Day-6                                                                              Day-7                                                                             Kl                                        __________________________________________________________________________    SA (12.6%)                                                                           3 0.3  0.6 1.3  1.7 2.3  2.7 4.4                                       Pyrocatechol                                                                         3 0.8  2.0 2.3  3.0 3.0  3.3 11.1                                      (12%)                                                                         NDGA (12%)                                                                           3 1.3  1.7 2.3  2.7 2.7  2.7 6.7                                       __________________________________________________________________________

Effects of Pyrocatechol, NDGA and SA on Cell Proliferation and ProteinSynthesis

The following experimental protocol was used to determine whetherpyrocatechol or SA had any effect on the rate of cell proliferation orprotein synthesis by cultured NHEK cells. A separate experiment wasconducted to determine if NDGA had any effect on cell proliferation.NHEK cells are grown in monolayers in 6 well plastic tissue cultureplates in serum free KGM Medium (Clonetics) until confluency ofapproximately 80% was achieved. To culture the cells, the plates wereplaced in a humidified, 5% CO₂ incubator at 37° C. To the cells wereadded various concentrations of either SA, pyrocatechol, or NDGA withappropriate controls, for 18 hours (overnight). Simultaneous with thistreatment, 1 μCi/ml each of ³⁵ S-methionine (protein precursor) and ³H-thymidine (DNA precursor) were added to the cells. For NDGA group, thecells were treated with ³ H-thymidine only. Following the treatmentphase, the cells were washed, harvested and counted in a liquidscintillation counter (triplicate wells/treatment group) to determinethe levels of incorporation of the radiolabeled precursors into cellularDNA and protein. The level of radioactivity recovered in the cells is ameasure of cells proliferation and protein synthesis.

The results of these experiments are shown in Tables 2, 3 and 4. As usedin Tables 2, 3 and 4, DPM means disintegrations per minute.

                  TABLE 2                                                         ______________________________________                                        Effects of SA on DNA and Protein Synthesis in NHEK Cells                                  .sup.3 H-Thymidine                                                                         .sup.3 H-Methionine                                  Dose        DPM ± SD  DPM ± SD                                          ______________________________________                                        SA 10 mM    26262 ± 18051                                                                            51611 ± 25175                                    SA 20 mM    3012 ± 1721                                                                             1767 ± 123                                        SA 30 mM    820 ± 7.0 1794 ± 113                                        EtOH 0.9%   264069 ± 34572                                                                          304993 ± 15662                                    (vehicle)                                                                     Untreated   3906020 ± 82954                                                                         377252 ± 15662                                    (control)                                                                     ______________________________________                                    

                  TABLE 3                                                         ______________________________________                                        Effects of Pyrocatechol on DNA and Protein Synthesis in NHEK                  Cells                                                                                       .sup.3 H-Thymidine                                                                         .sup.3 H-Methionine                                Dose          DPM ± SD  DPM ± SD                                        ______________________________________                                        Pyrocatechol 10 mM                                                                          3437 ± 4203                                                                             12375 ± 9863                                    Pyrocatechol 20 mM                                                                          890 ± 79  5928 ± 19                                       Pyrocatechol 30 mM                                                                          975 ± 123  7837 ± 21246                                   EtOH 0.9%     264069 ± 34572                                                                          304993 ± 21246                                  (vehicle)                                                                     Untreated     3906020 ± 82954                                                                         377252 ± 15662                                  (control)                                                                     ______________________________________                                    

                  TABLE 4                                                         ______________________________________                                        Effects of NDGA on DNA synthesis in NHEK cells                                              .sup.3 H-Thymidine                                              Dose          DPM ± SD                                                     ______________________________________                                        NDGA 20 mM    4,867 ± 247                                                  EtOH 0.9%     93,237 ± 19,382                                              (vehicle)                                                                     Untreated     203,520 ± 536                                                (control)                                                                     ______________________________________                                    

Both SA and pyrocatechol inhibit cell proliferation and proteinsynthesis in NHEK cells. Under the conditions of these analyses,pyrocatechol was more effective in blocking DNA and protein synthesisthan SA. These compounds exhibited strong dose-dependent inhibition ofDNA and protein synthesis (≧90% of control values). In a single doseexperiment, NDGA also showed strong inhibition of DNA synthesis in amanner similar to pyrocatechol and SA. To show that the effects ofpyrocatechol and SA were specific for DNA and protein synthesis and notdue to cell death, the viability of the treated cells was determined bya neutral red assay. The results of this assay showed that 80-95% of thetreated cells were viable. This activity is an important factor incorn/callus treatment (keratolysis) where hyperplastic growth of theskin is one of the observed pathological changes.

Effects of Pyrocatechol and SA on Intracellular Calcium Levels

An in vitro assay method was employed to measure calcium flux induced bythe compounds under investigation. Human squamous carcinoma cells (SCC-9cell line) were grown to confluent monolayers in 75 cm² tissue cultureflasks at 37° C. in a humidified, 5% CO₂ incubator. The cells in eachflask were cultured in 15 ml of complete medium, consisting of: Ham'sF12 Medium and Dulbecco's Modified Eagle's Medium mixed 1:1 (GibcoLaboratories, Grand Island, N.Y.), 10% fetal bovine serum (HycloneLaboratories, Logan, Utah), 0.4 μg/ml hydrocortisone, 2 mM L-glutamine,100 U/ml penicillin, 100 μg/ml streptomycin and 0.25 μg/ml amphotericinB (Sigma). On days that the calcium flux experiments were conducted thecell culture medium was removed and replaced with medium containing 8 μMIndo-1AM (Sigma) and 10 μ1/ml pluronic surfactant F-127 (Calbiochem, SanDiego, Calif.) and incubated for 1 hour at 37° C. The medium was removedand fresh tissue culture medium was added to the flask and the cellswere incubated at 37° C. for 1 hour. The cell monolayer was disruptedand the cells dispersed into a single cell suspension by removing thetissue culture medium, washing the monolayer with phosphate bufferedsaline (PBS), adding 2.5 ml of trypsin solution (0.5 mg/ml) (Sigma) andincubating for 10 minutes at 37° C. The cells were washed andresuspended in buffer containing 1.5 mM calcium. Test compoundsdissolved in 200 proof ethanol were added to cell aliquots and calciuminduced Indo-1 fluorescence was measured spectrophotometrically withexcitation at 355 nm and emission at 407.6 nm. The results of theexperiment are presented in Table 5

                  TABLE 5                                                         ______________________________________                                        Calcium Flux in SCC-9 Cells                                                   Compound       Dose    Relative Fluorescence                                  ______________________________________                                        (nontreated control)   0                                                      SA             176 μM                                                                             40.56                                                  Pyrocatechol   176 μM                                                                             8.82                                                   4BrA23187       19 μM                                                                             42.32                                                  (positive control)                                                            ______________________________________                                    

The relative levels of fluorescence (i.e., an indirect measure ofintracellular calcium concentration) above nontreated control levels,for cells treated with SA (176 μM) and calcium ionophore 4-Br-A23187 (19μM) were virtually identical at 40.56 and 42.32, respectively. Incomparison, the intracellular calcium concentration in cells treatedwith 176 μM pyrocatechol was significantly less (relativefluorescence=8.82). The results of this study indicate that SA andpyrocatechol are capable of inducing increased intracellular calciumflux in human keratinocytes (SCC-9 cells). Such activity indicates thatSA and pyrocatechol are useful as agents in causing enhancedkerotinocyte differentiation.

Effects of Pyrocatechol and SA on Ultrastructural Localization ofCalcium in Normal Human Skin

Pieces of full thickness, normal human skin were placed in organ culturewith an air/liquid interface at the level of the stratum corneum. Theorgan culture Medium was as follows: Dulbecco's Modified Eagle's Medium(DMEM); 100 μg/ml, hydrocortisone, hemisuccinate; 2 mM L-glutamine; 300U/ml penicillin; 30 mg/ml streptomycin; and 0.75 amphotericin B (Sigma).12.6% (weight/weight) of each test compound was dissolved in theflexible collodion/acetone vehicle described at Example E below of thisapplication and was applied topically to the skin pieces and incubatedfor 48 hours at 37° C. The skin pieces were then washed and prepared forobservation by transmission electron microscopy (TEM). Calcium wasprecipitated within the tissue by incubating the skin overnight in icecold fixative (at pH 7.4) consisting of: 2% glutaraldehyde, 2%formaldehyde, 90 mM potassium oxalate and 1.4% sucrose. In thepostfixative procedure the skin was incubated for 2 hours at 4° C. in 1%osmium tetroxide and 2% potassium pyroantimonate adjusted to pH 7.4 withacetic acid. The tissues were sectioned and lightly stained with uranylacetate and mounted on grids for examination using a JOEL 2000 TEM.

The results of this study are described just below.

In nontreated, control skin, the distribution of calcium within akeratinocyte in the stratum spinosum is as follows. Calcium isassociated in aggregates that are evenly dispersed throughout thenuclear euchromatin. Calcium is also readily observed in the cytoplasmof this cell and extracellular compartment. At the level of the stratumgranulosum, high concentrations of calcium are seen in the extracellularspace along the cell borders and in association with the desmosomes. Incontrast, calcium is not detected, by this method, in the extracellularcompartment, in association with desmosomes or in the cytoplasm ofkeratinocytes of skin treated with pyrocatechol. High concentrations ofcalcium are distributed throughout the nuclear heterochromatin ofkeratinocytes from pyrocatechol treated skin. Evidence of desmosomedegradation is also observed in this tissue section. In the stratumspinosum of skin treated with SA the distribution of calcium is similar,but not identical, to that observed in pyrocatechol treated skin. Forexample, there is very little calcium seen in either the extracellularcompartment or associated with desmosomes. Unlike the effect ofpyrocatechol, SA treatment did not cause degradation of desmosomes.Calcium in the nuclei of keratinocytes in skin treated with SA is highlyconcentrated within the heterochromatin. In one section of skin treatedwith SA, two keratinocytes can be observed. In one keratinocyte calciumis not detected in the euchromatin, in the other keratinocyte calcium isthinly dispersed in the euchromatin. Collectively, these datademonstrate the variable effects of these compounds on calciumlocalization within the skin. Changes in calcium distribution from theextracellular compartment to the nuclear heterochromatin are linked toenhanced keratinocyte differentiation and desquamation and these studiesclearly show that the effect of pyrocatechol≧SA in causing thesechanges.

Effect of Pyrocatechol, NDGA and SA on the Expression and Distributionof Desmosomal Protein, Desmoglein, in the Epedermis of Organ CulturedNormal Human Skin

Immunoperoxidase staining was employed to detect desmoglein (DG)expression in organ cultured skin treated with pyrocatechol, NDGA andSA. Pieces of full thickness normal human skin were placed in organculture at an air/liquid interface at about the level of the stratumcorneum. Test compounds were added to the tissue culture medium at afinal concentration of 15 mM. An equal volume of ethanol, which was usedas the solvent for the stock test compounds, was added to the medium ofthe control cultures. The skin pieces were incubated for 24 hours at 37°C. before they were prepared for frozen sectioning. After removal fromorgan culture the pieces of skin were fixed in O.C.T. Embedding Medium(Tissue-Tek, Miles Laboratories, Inc., Elkhart, Ind.) and frozen inliquid nitrogen. Frozen sections of O.C.T. fixed tissues were cut on acryostat set to 5 micron thickness and the sections were placed on glassslides. The tissue sections were rehydrated with PBS for 10 minutes atroom temperature. To reduce nonspecific antibody binding the tissuesections were covered with normal goat serum (Accurate Chemical andScientific, Corp., Westbury, N.Y.), diluted 1:20 with PBS, and incubatedin a humidified chamber at room temperature for 20 minutes. The tissueswere washed with PBS, covered with 100 μl of primary antibody solutionand incubated for 2 hours at room temperature. Primary antibodies usedin this study were antidesmoglein (Boehringer Mannheim Biochemicals,Indianapolis, Ind.), a mouse monoclonal IgG 2b antibody raised againsthuman 165 kD DG protein, and normal, nonimmune mouse serum (Accurate)that served as a nonspecific binding control. The primary antibodieswere diluted in PBS at 1:100 for antiDG and 1:500 for normal mouse serumfor tissue binding. After binding of the primary antibodies wascompleted, the tissue sections were washed 3× with excess amounts ofPBS. The sections were blotted dry on a paper towel, covered with 100 μ1of a secondary antibody, biotin-conjugated goat antimouse IgG (Accurate)diluted 1:100 with PBS, and incubated 20 minutes at room temperature.The sections were washed 3× in excess PBS. They were covered withhorseradish peroxidase conjugated streptavidin (Accurate) that wasdiluted 1:100 in PBS and incubated for 20 minutes at room temperature.The tissue sections were washed 3× in excess amounts of PBS andincubated 3 minutes in AEC solution, consisting of: 20 mg3-amino-9-ethylcarbazole dissolved in 5 ml dimethylsulfoxide and mixedwith 50 μl of hydrogen peroxide (30% solution) in 20 mM acetate bufferat pH 5.2, which results in a reddish precipitate at sites ofperoxidase-antibody binding. After washing off the AEC solution withPBS, the tissues were lightly counterstained with hemotoxylin. Thesections were washed with PBS and mounted in glycerol under a coverglass for light microscopic examination. The results of this study aredescribed below.

DG is expressed throughout the stratum spinosum layer of control skin,as well as skin treated with SA. In contrast, pyrocatechol treated skinshowed no detectable staining for DG protein. This indicates thatpyrocatechol is useful as an agent in inducing desquamation. NDGAdecreased DG expression determined by stain development to a greaterlevel than SA but not as great as pyrocatechol. NDGA is also a usefulagent in inducing desquamation.

These observations are consistent with the observed ultrastructuralchanges to desmosomes in TEM photomicrographs.

Effects of Pyrocatechol, NDGA and SA on Cross-linked Cellular EnvelopeFormation

An in vitro assay method was used to measure cellular envelope (CE)formation induced in cultured keratinocytes by the three compounds underinvestigation. Human squamous carcinoma cells (SCC-9 cell line) weregrown to confluent monolayers in 25 cm² plastic tissue culture flasks at37° C. in a humidified, 5% CO₂ incubator. The cells in each flask werecultured in 10 ml of complete medium, consisting of: Ham's F12 Mediumand Dulbecco's Modified Eagle's Medium mixed 1:1 (Gibco), 10% fetalbovine serum (Hyclone), 0.4 μg/ml hydrocortisone, 2 mM L-glutamine, 100U/ml penicillin, 100 μg/ml streptomycin and 0.25 μg/ml amphotericin B(Sigma). On days that the CE induction experiments were performed thecell culture medium was removed and the cell monolayer was washed with20 ml of PBS. The cell monolayers were then covered with 5 ml of Hank'sbalanced salt solution formulated with 2 mM calcium. A fixed volume fromstock solutions were added to each flask to give final concentrations ofcalcium ionophore A23187 (100 μM), pyrocatechol (15 mM), NDGA (15 mM)and SA (15 mM) (Sigma). All experimental groups were run in triplicate.An equal volume of ethanol, the solvent used to prepare the stocksolutions, was added to the flasks designated as controls. The cellswere then incubated for 6 hours in a humidified, 5% CO₂ incubator at 37°C. A rubber policeman was used to scrape the cells off the surface ofthe flasks. The cells were transferred to 15 ml plastic centrifuge tubesand pelleted by centrifugation at 800×g for 10 minutes. The supernatantswere removed and the cells were resuspended in 1 ml of PBS containing 2%sodium dodecyl sulfate (SDS), 20 mM dithiothreitol and 30 μg DNAase(Sigma) to extract the CE. The CE were dispersed into suspension byplacing the tubes in a waterbath sonicator for 10 minutes. They werethen placed in a drying oven with the ambient air temperature at 100° C.for 10 minutes. The CE were then sonicated for 10 minutes, an additional30 μg of DNAase was added and the CE were allowed to cool at roomtemperature for 10 minutes. The contents of the 15 ml centrifuge tubeswere transferred to plastic 1.8 ml microfuge tubes using disposable,glass pasture pipets. The CE were pelletted in a microcentrifuge at1000×g for 10 minutes at 25° C. The supernatants were discarded and theCE were resuspended in 1 ml of 0.1% SDS in PBS and pelleted at 1000×gfor 10 minutes at 25° C. The CE were resuspended in 1 ml of 0.1% SDS inPBS and analyzed by spectrophotometry. Absorption (i.e., light scatter)measurements were made at 340 nm.

The results of this study are presented in Table 6.

                  TABLE 6                                                         ______________________________________                                        Cellular Envelope Formation in SCC-9 Cells                                                           Absorbance @ 340 nm                                    Compound      Dose     (mean ± SEM × 10.sup.-4)                      ______________________________________                                        A23187        100 μM                                                                               922 ± 119                                          Pyrocatechol  15 mM    9316 ± 323                                          SA            15 mM    566 ± 98                                            Et OH Control          328 ± 14                                            ______________________________________                                    

The data are given as the mean±S.E.M. absorption value for each testgroup. As can be seen, CE were induced by treating the SCC-9 cells witheither ionophore A23187 or SA. About twice as many CE were induced byionophore as were induced by SA. In comparison, about 20 times more CEwere induced by pyrocatechol. The studies for NDGA were evaluated usingthe above method and then counting all cell envelopes in ahemocytometer. The results are as follows:

    ______________________________________                                                       Concentration of cell                                          Compound       envelopes in (CE)/ml                                           ______________________________________                                        pyrocatechol (15 mM)                                                                         3.2 × 10.sup.5                                           SA (15 mM)     0.2 × 10.sup.5                                           NDGA (15 mM)   1.0 × 10.sup.5                                           ______________________________________                                    

These data indicate that pyrocatechol and NDGA are both potentstimulators of CE formation in SCC-9 cells compared to the activities ofcalcium ionophore A23187 and SA. NDGA produced about 5 times the numberof CE as SA. A23187 is the antibiotic calcimycin with the formula C₂₉H₃₇ N₃ O₆ which is described on page 249, entry 1639 of Merck Index,11th Edition, 1989, S. Budavari, Editor, Merck and Company, Rahway,N.J., and 4BrA23187 is a halogenated analog of A23187, available fromSigna Chemical Co. St. Louis, Mo.

Effect of Pyrocatechol and SA on Lipid Synthesis by Keratinocytes

It has been reported that the amounts of various classes of lipidschange as keratinocytes differentiate through the various layers of theepidermis (Lampe, M. A. , M. L. Williams and P. M. Elias. 1983. Humanepidermal lipids: characterization and modulations duringdifferentiation. J Lipid Res 24:133-140). The distribution of lipids inthe different epidermal layers of normal skin is shown in Table 6. Themost significant change is the decrease in polar lipids and increase inneutral lipids that occurs as the keratinocytes progress from the viablecell layers into the stratum corneum. This indicates that a shift frompolar lipid to neutral lipid synthesis is associated withdifferentiation of keratinocytes to corneocytes. The followingexperiments were thus conducted to determine whether the two compoundstested effected keratinocyte lipid synthesis.

Human squamous carcinoma cells (SCC-9 cell line) were grown to confluentmonolayers in 75 cm² tissue culture flasks at 37° C. in a humidified, 5%CO₂ incubator. The cells in each flask were cultured in 15 ml ofcomplete medium, consisting of: Ham's F12 Medium and Dulbecco's ModifiedEagle's Medium mixed 1:1 (Gibco), 10% fetal bovine serum (Hyclone), 0.4μg/ml hydrocortisone, 2 mM L-glutamine, 100 U/ml penicillin, 100 μg/mlstreptomycin and 0.25 μg/ml amphotericin B (Sigma). On the day of theexperiment the tissue culture medium was removed and replaced with freshmedium containing one of the test compounds. Cells were incubated 24hours and then scraped from the flask using a rubber policeman. Thecells were pelletted by centrifugation at 800×g for 10 minutes. The cellpellet was freeze-dried in a lyophilizer overnight and a dry weight wasdetermined on the material.

The Bligh-Dyer procedure (Bligh, E. G. and W. J. Dyer. 1959. A rapidmethod of total lipid extraction and purification. Can. J. Biochem.Physiol. 37:911-917), was employed to extract the lipid from thefreeze-dried cellular material. More specifically, the procedure usedwas as follows: the dried material was suspended in 7.5 ml Bligh-DyerSolution (Chloroform/Methanol/Water at 1:2:0.8 by volume) to dissolvethe lipid. The lipid extraction was performed so that the dry weight ofthe material did not exceed 30 mg/ml of Bligh-Dyer Solution. Thematerial remained suspended in the Bligh-Dyer Solution overnight at 4°C. The material was then homogenized using a Dounce tissue homogenizerwith a tight fitting piston. The solution containing the dissolved lipidwas combined with an equal amount of Bligh-Dyer Solution that was usedto rinse the homogenizer. The combined solutions were then pouredthrough a shark skin filter. The filtrate (organic solution containingthe lipids) was dried down under a continuous flow of nitrogen gas andthen redissolved in 7.5 ml Bligh-Dyer Solution. To that solution 2 ml ofwater and 2 ml of chloroform were added. The tube was vortexed to mixthe solution and then centrifuged at 1000×g for 10 minutes to separatethe organic and aqueous phases. The bottom, organic, chloroform phasewas removed and transferred to a separate centrifuge tube. An equalvolume of wash solution (Chloroform/Methanol/Water at 1:1:0.9 by volume)was added to the organic phase. The tube was vortexed and centrifuged asabove. The bottom phase was collected and the organic solvent wasevaporated under nitrogen gas. The dried lipid was dissolved in 0.5 mlwarm absolute ethanol and transferred, along with an equal volume ofethanol that was used to rinse the tube, to a microfuge tube. Thecontents of the microfuge tube were blown dry under nitrogen gas. Thedried sample was dissolved in 500 μl of Chloroform/Methanol at 2:1 byvolume.

The dry weight of the lipid extract was determined by placing 25 μl ofthe sample in a small preweighed aluminum pan. The weighing pan wasplaced on a hot plate set on a low temperature to evaporate the solvent.The pan was cooled for about 15 seconds and then weighed on a Cahn C31Microbalance. The average weights were determined for each sample bymeasuring triplicates and the concentration of each sample wascalculated.

High-performance thin-layer chromatography (HPTLC) was employed toseparate the lipids for analysis. The sample concentration was adjustedto 0.17 μg/ml by adding an appropriate volume of absolute ethanol. AnAnaltech Silica Gel G 20×20 cm² HPTLC plate was "cleaned" by running thesystem I (Methanol/Chloroform/Water at 20:95:1 by volume) solvent to thetop of the plate. The plate was air dried and then heat activated in adrying oven at 110° C. for 30 minutes. Samples were applied 1.5 cm fromthe bottom of the plate in 4 mm wide bands using a Camag LinomatAutospotter. The plate was developed in solvent system I. The solventfront was allowed to migrate 6 cm from the origin. The plate was airdried and developed again in solvent system I. The plate was air driedand then developed in solvent system II(n-Hexane/Diethyl Ether/GlacialAcetic Acid at 8:2:1 by volume). The solvent front was allowed tomigrate 8.5 cm above the origin. The plate was air dried and thendeveloped in solvent system III (Petroleum Ether). The solvent front wasallowed to migrate 13 cm above the origin. The plate was air driedovernight at room temperature and then immersed for 20 seconds in asolution of 10% (w/v) cupric sulfate hydrate and 8% (w/v) phosphoricacid in water. The plate was drained on a paper towel and "charred" on aheat block at 180° C. for 1 hour. The lipid bands were quantitated,against a set of standards that were run on each plate, by scanningdensitometry using a Shimadzu CS9000U, Dual-wavelength, Flying SpotScanner.

The results of these analyses are presented in Table 8. Compared to thenontreated, control cells, there was a significant reduction in polarlipid, cholesterol sulfate and sphingolipids with increased amounts ofneutral lipids synthesized by cells treated with SA. Pyrocatechol alsohad a marked effect on lipid synthesis causing a complete reduction ofsphingolipids, a partial reduction in neutral lipids and cholesterolsulfate and a significant increase in polar lipids. Although thecompounds had distinct effects on SCC-9 cell lipid synthesis, in vitro,only SA appeared to induce a shift in lipid metabolism from that ofviable keratinocytes to that of comeocytes. Nevertheless, in view of allof the test results contained herein, Pyrocatechol has been demonstratedto be an active keratolytic agent.

                  TABLE 7                                                         ______________________________________                                        Variations in Lipid Composition During Human Epidermal                        Differentiation and Cornification.                                            Lipid Fraction                                                                           S. Basal/Spinosum                                                                          S. Granulosum                                                                            S. Corneum                                 ______________________________________                                        Polar Lipids                                                                             44.5 ± 3.4*                                                                             25.3 ± 2.6                                                                            4.9 ± 1.6                               Cholesterol Sulfate                                                                      2.4 ± 0.5  5.5 ± 1.3                                                                            1.5 ± 0.2                               Sphingolipids                                                                            7.3 ± 1.0 11.7 ± 2.7                                                                            18.1 ± 2.8                              Neutral Lipids                                                                           51.0 ± 4.5                                                                              56.5 ± 2.8                                                                            77.7 ± 5.6                              ______________________________________                                         *Each datum is presented as percent of total lipid extracted from normal      abdominal skin.                                                          

The Table is adapted from: Lampe, M. A., M. L. Williams and P. M. Elias.1983. Human epidermal lipids: characterization and modulations duringdifferentiation. J Lipid Res 24:133-140.

                  TABLE 8                                                         ______________________________________                                        Effects of Pyrocatechol and SA on the Lipid Composition of                    SCC-9 cells.                                                                          Polar    Cholesterol                                                  Treatment.sup.1                                                                       Lipids   Sulfate   Sphingolipids                                                                          Neutral Lipids                            ______________________________________                                        Control 27.65.sup.2                                                                            22.95     11.95    37.45                                     SA      10.90    15.24     2.37     71.49                                     Pyrocatechol                                                                          56.30    9.80      0.00     33.90                                     ______________________________________                                         .sup.1 Final concentrations of the designated compounds in the tissue         culture medium were: SA, 15 mM and Pyrocatechol, 15 mM.                       .sup.2 Each datum is presented as percent of total lipid extracted from       each test group.                                                         

Further examples of the invention are as follows:

    ______________________________________                                        Example A:                                                                                   Percentage of formula                                          Component      % (weight/weight)                                              ______________________________________                                        Olivetol       12                                                             Dipropylene Glycol                                                                           88                                                             ______________________________________                                    

The composition containing 12% (weight/weight) olivetol can be made asfollows:

1. The olivetol is placed in a suitable vessel.

2. Propylene glycol is added to the above vessel and the resultingmixture is stirred until a solution was formed.

3. The resulting composition is transferred to appropriate containersfor storage at room temperature (22°-27° C).

    ______________________________________                                        Example B:                                                                                   Percentage of formula %                                        Component      (weight/weight)                                                ______________________________________                                        Hydroquinone   12                                                             Acetone        10                                                             Dipropylene Glycol                                                                           78                                                             ______________________________________                                    

The above composition containing 12% (weight/weight) hydroquinone can bemade as follows:

1. The hydroquinone is placed in a suitable vessel.

2. Acetone is added to the hydroquinone in the vessel and mixed until asolution was formed.

3. To the mixture formed in step 2, flexible collodion is added to reachthe desired final volume and mixed until the entire contents arethoroughly mixed.

4. The composition is transferred to appropriate containers for storageat room temperature (22°-27° C).

    ______________________________________                                        Example C:                                                                    Component     Percentage of formula % (weight/weight)                         ______________________________________                                        Pyrocatechol  6.0                                                             Balsam fir (Oregon)                                                                         5.0                                                             β-Carotene                                                                             0.05                                                            Acetone       10.0                                                            Flexible collodion (USP)                                                                    qs 100                                                          ______________________________________                                    

The above composition containing 6% (weight/weight) pyrocatechol can beprepared as follows:

1. Balsam fir (Oregon) and (β-carotene are mixed with acetone in asuitable vessel until thoroughly mixed.

2. Pyrocatechol is added to the mixture formed in step 1 and mixed untildissolved.

3. Flexible collodion is added to the above mixture and stirred untilcompletely mixed.

4. The composition is then transferred to appropriate containers andstored at room temperature 22° C.-27° C.

Preferred ingredients of compositions of the invention fall within thefollowing ranges:

    ______________________________________                                                           Percentage of Formula %                                    Component          (weight/weight))                                           ______________________________________                                        Pyrocatechol or NDGA                                                                              3-24                                                      Balsam Fir (Oregon)                                                                              2-5                                                        β-Carotene, 22% in Vegetable Oil                                                            0.01-0.05                                                  Acetone             5-15                                                      Flexible Collodion, USP                                                                          66-90                                                      ______________________________________                                    

More preferred ingredients of compositions of the invention fall withinthe following ranges:

    ______________________________________                                                          Percentage of formula %                                     Component         (weight/weight)                                             ______________________________________                                        NDGA               6-20                                                       Ethyl Alcohol (190 proof, USP)                                                                  30-60                                                       Arlasolve 200 Liquid                                                                            15-50                                                       Klucel HXF-NF     0.3-0.7                                                     ______________________________________                                    

A most preferred composition of the invention is

    ______________________________________                                                          Percentage of formula %                                     Component         (weight/weight)                                             ______________________________________                                        NDGA              12.00                                                       Ethyl Alcohol (190 proof, USP)                                                                  48.50                                                       Arlasolve 200 Liquid                                                                            39.00                                                       Klucel HXF-NF     00.50                                                       ______________________________________                                    

A preferred composition of the invention is

    ______________________________________                                        Example D                                                                                         Percentage of formula %                                   Component           (weight/weight)                                           ______________________________________                                        Pyrocatechol        12.00                                                     Acetone             10.00                                                     Flexible collodion (USP)                                                                          72.95                                                     Balsam fir (Oregon) 5.00                                                      β-Carotene HSE 22% in vegetable oil                                                          0.05                                                      ______________________________________                                    

It was made as follows:

1. Pyrocatechol and acetone were mixed in a suitable tank equipped withan explosion proof mixer.

2. Flexible collodion was added and mixed until the pyrocatechol was incomplete solution.

3. Balsam Fir Oregon and β-Carotene were added and mixed well until aclear uniform mixture was obtained.

4. The mixture was stored in appropriate containers at room temperature,22° C.-27° C.

Another preferred composition of the invention is

    ______________________________________                                        Example E                                                                                         Percentage of formula %                                   Component           (weight/weight)                                           ______________________________________                                        NDGA                12.00                                                     Acetone             10.00                                                     Flexible collodion (USP)                                                                          72.95                                                     Balsam fir (Oregon) 5.00                                                      β-Carotene HSE 22% in vegetable oil                                                          0.05                                                      ______________________________________                                    

It was made in the same manner as Example E above except that NDGA wasused in place of pyrocatechol.

The most preferred composition of the invention is

    ______________________________________                                        Example F                                                                                       Percentage of formula %                                     Component         (weight/weight)                                             ______________________________________                                        Part A:                                                                       NDGA              12.00                                                       Ethyl Alcohol (190 proof, USP)                                                                  48.50                                                       Part B:                                                                       Arlasolve 200 Liquid.sup.1                                                                      39.00                                                       Klucel HXF-NF.sup.2                                                                             00.50                                                       ______________________________________                                         .sup.1 Arlasolve 200 Liquid is Arlasolve 200LIsoceteth-20 which is a          polyethylene glycol ether of isocetyl alcohol that conforms generally to      the formula C.sub.16 H.sub.33 (OCH.sub.2 CH.sub.2).sub.n OH where n has a     average value of 20. Other names for this material are PEG20 Isocetyl         Ether: Polyethylene Glycol 1000 Isocetyl Ether; and Polyoxyethylene (20)      Isocetyl Ether.                                                               .sup.2 Klucel HXFNF which is KlucelHydroxypropylcellulose which is a          propylene glycol ether of cellulose. Another name for this material is        2hydroxypropyl ether cellulose.                                          

Procedure

1. NDGA was added to the alcohol of Part A and mixed until dissolved.

2. In a container large enough to contain the entire batch, thearlasolve of part B was transferred, and the klucel of part B was addedand mixed until well dispersed.

3. While mixing the mixture of step 2, the mixture of step 1 was addedand mixed until the gel was uniform and klucel was completely hydrated.

4. The resulting material was stored in a closed container.

What is claimed is:
 1. A topical composition comprising a about 2 toabout 64% of a compound selected from the group consisting ofhydroquinone; olivetol, pyrocatechol and nordihydroguaiaretic acid and apharmaceutically acceptable carrier material wherein said carriermaterial is flexible collodion.
 2. A composition in accordance withclaim 1 which further comprises an enhancer selected from the groupconsisting of salicylic acid, erucic acid, and propionic acid.
 3. Amethod for the treatment of hyperkeratinizing and hyperproliferativeskin diseases and conditions which comprises topically administering atherapeutically effective amount of a composition defined in claim
 1. 4.A method for removing corns, calluses and warts which comprisestopically administering a therapeutically effective amount of acomposition defined in claim
 1. 5. A method in accordance with claim 1wherein the compound is pyrocatechol.
 6. A method in accordance withclaim 1 wherein the compound is nordihydroguaiaretic acid.
 7. A topicalcomposition comprising on a weight/weight basis:

    ______________________________________                                        Percentage of Formula %                                                       (weight/weight)                                                                             Component                                                       ______________________________________                                         3-24         Pyrocatechol or Nordihydroguaiaretic acid                       2-5           Balsam Fir (Oregon)                                             0.01-0.05     β-Carotene, 22% in Vegetable Oil                            5-15         Acetone                                                         66-90         Flexible Collodion, USP                                         ______________________________________                                    


8. A topical composition in accordance with claim 7 comprising:

    ______________________________________                                        Percentage of Formula % (weight/weight)                                                            Component                                                ______________________________________                                        6.0                  Pyrocatechol                                             5.0                  Balsam Fir (Oregon)                                      0.05                 β-Carotene                                          10.0                 Acetone                                                  qs 100               Flexible Collodion, USP                                  ______________________________________                                    


9. A topical composition in accordance with claim 7 comprising:

    ______________________________________                                        Percentage of Formula %                                                       (weight/weight) Component                                                     ______________________________________                                        12.00           Pyrocatechol                                                  10.00           Acetone                                                       72.95           Flexible collodion (USP)                                      5.00            Balsam fir (Oregon)                                           0.05            β-Carotene HSE 22% in vegetable oil                      ______________________________________                                    


10. A topical composition in accordance with claim 7 comprising:

    ______________________________________                                        Percentage of Formula %                                                       (weight/weight) Component                                                     ______________________________________                                        12.00           Nordihydroguairaretic acid                                    10.00           Acetone                                                       72.95           Flexible collodion (USP)                                      5.00            Balsam fir (Oregon)                                           0.05            β-Carotene HSE 22% in vegetable oil                      ______________________________________                                    


11. A topical composition comprising on a weight/weight basis:

    ______________________________________                                        Percentage of Formula %                                                       (weight/weight)  Component                                                    ______________________________________                                         6-20            Nordihydroguaiaretic acid                                    30-60            Ethyl Alcohol (190 proof, USP)                               15-50            Arlasolve 200 Liquid.sup.1                                   0.03-0.7         Klucel HXF-NF.sup.2                                          ______________________________________                                    


12. A topical composition in accordance with claim 11 comprising:

    ______________________________________                                        Percentage of Formula %                                                       (weight/weight)  Component                                                    ______________________________________                                        12.00            Nordihydroguaiaretic acid                                    48.50            Ethyl Alcohol (190 proof, USP)                               39.00            Arlasolve 200 Liquid.sup.1                                   00.50            Klucel HXF-NF.sup.2                                          ______________________________________                                    